A duplex real-time PCR assay for the detection and differentiation of Leishmania infantum and Leishmania tarentolae in vectors and potential reservoir hosts

Leishmanioses are vector-borne diseases, some of zoonotic concern, transmitted by phlebotomine sand flies (Diptera, Psychodidae). Reports of reptile-associated Leishmania tarentolae in humans, and of Leishmania infantum in Sergentomyia minuta sand flies prompted the development of an internal transcribed spacer 1-based duplex quantitative real-time PCR (dqPCR) to detect and differentiate these Leishmania spp. The specificity of dqPCR was assessed by processing DNA samples from Phlebotomus spp. (n = 188) and Se. minuta (n = 171) and from tissues (i.e., heart, liver, muscle, lungs, spleen, kidney, eggs) of Podarcis siculus (n = 4) and Tarentola mauritanica (n = 3). The analytical sensitivity of the dqPCR, assessed using 10-fold serial dilutions of DNA from Leishmania spp. and spiked DNA samples from lizards, was 2.3 × 10-7 ng/2 µl for L. infantum and 2.1 × 10-7 ng/2 µl for L. tarentolae. The dqPCR detected up to 4.3 × 10-6 ng/2 µl of L. infantum and up to 4.9 × 10-7 ng/2 µl of L. tarentolae. Of the 359 phlebotomine sand flies tested, five (3.6%) and two (1.4%) Ph. perniciosus scored positive for L. infantum and L. tarentolae, respectively. Similarly, of 171 Se. minuta, 56 (32.7%) and six (3.5%) scored positive for L. tarentolae and L. infantum, respectively. Co-infection was detected in two Se. minuta (1.2%). Out of seven reptiles tested, four P. siculus were positive for L. tarentolae. This new dqPCR may improve the diagnosis of L. infantum and L. tarentolae and aid to assess the role of lizards as reservoirs and of Se. minuta as vector, for these Leishmania spp.